With no vaccine in sight, there is an urgent public health need to develop an effective topical microbicide that can reduce the number of new HIV-1 infections in women. The potential role of virus-cell fusion inhibitor-based microbicides in preventing mucosal transmission of HIV-1 has been clearly identified. However, none of the reported gp41 fusion inhibitors has made significant progress toward clinical trials. HIV-1 infection requires fusion of the viral and cellular membranes, driven by association of two heptad-repeat regions in the gp41 ectodomain to form a highly stable six-helix bundle structure. Whereas this postfusion motif comprising native N36 and C34 peptides has no inhibitory activity, the isolated peptides inhibit HIV-1 entry by binding to their cognate sites on gp41. Our goal in this MIP VI application is to develop an inexpensive, potent, structured 'pro- drug' form of the N- and C-peptide fusion inhibitors that exhibits significant microbicidal activity upon use in situ. Our development effort will be based on preliminary data obtained with a truncated six-helix bundle that inhibits in vitro infection by primary HIV-1 isolates with low nanomolar IC50 values. We propose a comprehensive, interdisciplinary approach that combines high-resolution structural determination, recombinant protein production and mutagenic analyses, virology, and animal model efficacy studies. In this project we seek to conduct in vitro and in vivo preclinical and animal model-based research intended to facilitate the development of new HIV-1 gp41 peptide fusion inhibitor as a practical microbicide. The Specific Aims are: 1. To optimize and identify HIV-1 peptide fusion inhibitors for development as a vaginal microbicide. (a) To identify and incorporate specific amino-acid residue substitutions that optimize both potency and solubility of fusion inhibitor peptides. (b) To develop and optimize robust procedures for the large-scale bacterial expression and purification of select fusion inhibitor peptides. (c) Investigate the mechanisms of resistance to peptide inhibitors so as to avoid eliciting resistance. 2. To characterize the specificity, potency and toxicity of optimized peptide fusion inhibitors and their in vitro synergistic interactions with the CCR5 inhibitor CMPD167 and the entry inhibitor BMS-378806. (a) Determine the virucidal activity of optimized fusion inhibitor peptides against a diverse set of primary HIV-1 isolates. (b) Evaluate their toxicity, immunogenicity and drug stability in the rabbit model. (c) Study antiviral synergy in vitro in order to make rational predictions for lead inhibitor combinations for in vivo efficacy testing. 3. To test the effectiveness of the fusion inhibitor peptides to protect against mucosal HIV-1 infection. (a) Characterize the specificity and potency of effective peptide inhibitors in an in vitro model of HIV-1 infection of human cervical and vaginal tissue. (b) Use the NOD/SCID-hu BLT mouse vaginal transmission model to assess the in vivo potency and breadth of activity of highly effective peptide inhibitors alone and in combination with the small-molecule CCR5 inhibitor CMPD167 and the small-molecule entry inhibitor BMS-378806. 1